Danggui Buxue Tang (DBT) is a simple decoction, having about 800 years of usage in China to treat menopausal irregularity in women, which contains two herbs: Radix Astragali (Huangqi) and Radix Angelicae Sinensis (Danggui). Traditionally, boiling water has been used for preparing DBT; however, the optimized conditions of extraction have not yet been determined. Here, the amounts of Radix Astragali-derived astragaloside IV, calycosin, formononetin, and Radix Angelicae Sinensis-derived ferulic acid and ligustilide were determined in DBT, which were extracted according to an orthogonal array experimental design having three variable parameters: extraction time, extraction volume and number of repeats of the extraction. Our results suggest that extraction time and number of repeats of the extraction are two crucial factors, while extraction volume is a subordinate factor. The optimized conditions for extraction were therefore established. Besides the chemical composition, the estrogenic and anti-platelet aggregation activities of DBT were determined in different groups of the extraction, and the results of bioassays were in line with the amounts of the analyzed chemical ingredients. The results provide a model system for establishing the quality assurance of the herbal preparation.

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Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the general phenylpropanoid pathway leading to the production of phenolic compounds with a significant range of biological function. A PAL gene we designated gPAL1, cloned from tobacco, consists of two exons separated by an intron of 1932 bp. Exon I, 398 bp, and exon II, 1747 bp, together encode a polypeptide of 715 amino acids. A putative TATA box and polyadenylation signal are found 144 bp upstream of the initiation codon and 193 bp downstream from the stop codon, respectively. Using various parts of gPAL1 as probes, genomic Southern blots indicated the presence of a small family of PAL genes in the tobacco genome that can be divided into two distinct subfamilies, one consisting of pal1 and pal2 and another of pal3 and pal4. Comparative genomic blot analysis of progenitor species (Nicotiana tomentosiformis and N. sylvestris) indicated that each species contains one PAL gene from each of the subfamilies, suggesting that pal1 and pal3 (or pal2 and pal4) diverged prior to the evolution of N. tabacum. Expression of the PAL gene family was examined using RNA gel blots. PAL transcript levels were significantly higher in flowers and roots than in leaves and stems of mature plants. PAL transcripts accumulate differentially during flower and leaf maturation in that mRNA levels decline during flower maturation but increase during leaf maturation. In leaves, PAL transcripts rapidly accumulated afer wounding.